Plant Cell Reports

Plant Cell Rcports (1994) 14:171-174

,~ Springer-Verlag 1994

Somatic embryogenesis and plant regeneration from leaf-derived cell suspension of a mature t r e e Thevetia peruviana L. Abha Sharma 1 and Anjani Kumar 2 Department of Botany School of Life Sciences, North-Eastern Hill University, Shillong 793 014, India 2 Biotech Division, Feathers Farms Private Ltd., Orchid House, Bivar Road, P.O. Box No. 23, Shillong 793 001, India Rcccived 26 October 1993/Rcvised vcrsion receivcd 4 August 1994 - Communicated by G.C. Phillips

ABSTRACT Cell suspension cultures, w h i c h retained embryogenic potential for almost 2 years, w e r e established from young, expanding, j u v e n i l e leaves o f a mature Thevetia peruviana L. tree. CaUi were obtained by culturing y o u n g leaf discs on M S m e d i u m supplemented with 2 mg/L 2, 4-dichlorophenoxyacetic acid (2, 4-D) and 0.1 m g / L kinetin. Suspension cultures were initiated by transfer of calli to liquid m e d i u m containing 1 m g / L 2,4-D + 0.1 m g / L kinetin, and the cultures w e r e maintained by subculturing to fresh m e d i u m at 2 week intervals. E m b r y o g e n i e f r e q u e n c y o f cell aggregates was more than 8 0 % w h e n plated on semi-solid m e d i u m containing 0.1 mg/L 2, 4 - D and 2 m g / L 2-isopentenyladenine (2-iP). Cell aggregates with developing embryos were transferred to fresh m e d i u m lacking growth regulators for embryo maturation. Early embryo development was synchronous and a large n u m b e r o f somatic embryos were p r o d u c e d These somatic e m b r y o s developed into plantlets upon subsequent transfer to modified half-strength M S medium. M o r e than 200 green and rooted plants, at an average o f 80 plants per 100 m g o f embryogenic callus, were obtained with 6 0 % survival under glass house conditions.

ABBREVIATIONS 2, 4-D - 2 4-dichlorophenoxyacetic acid; 2-iP - 2-isopentenyladenine; I A A - hMole - 3 - acetic acid; K N - Kinetin; M S - Murashige and Skoog (1962) basal medium: N A A - 1 Napthalene acetic acid.

INTRODUCTION Thevetia peruviana L (Apocyanaceae) is a plant o f medicinal importance due to the presence o f cardioactive c o m p o u n d s in its latex. O f all the Thevetia glycosides, peruvoside is the most important and a substantial amount o f w o r k has been done on its pharmacology, toxicology and clinical aspects. Peruvoside has a higher therapeutic index than digoxin Correspondence to." A. Kumar

and is being marketed in G e r m a n y under the trade name ' Endocordin' (Bhakuni 1990). Thevetin, another cardioactive glucoside, produces theverisin, upon hydrolysis. Both thevetin and theverisin have digitalis-like cardiotonic effects, indicating their possible clinical applications. Efficient plant regeneration f r o m cultured cells/tissue is a prerequisite for in vitro manipulation (Eapen and George 1989; Baker and Wetzstein 1992). Somatic embryos, often being unicelhdar ha origin (Haccius 1978; Nagmani et al 1987), are well suited for mutant selection and genetic transformation. H o w e v e r , in vitro somatic embryogenesis has been reported only in a few h a r d w o o d species (yon Arnold 1988). Isolation of resistance mutants ha trees has lagged because of a lack o f selective systems. We reported earlier organogenesis ( K u m a r 1992a) and somatic embryogenesis ( K u m a r 1992b) f r o m callus of T. peruviana. Here we report establishment o f e m b r y o g e n i c cell suspension cultures and long term plantlet regeneration from this species.

MATERIALS AND METHODS Young leaves, collected from a 12-15 year old Thevetia peruviana L. tree, were agitated with Teepol solution (detergent) containing a few &ops of Tween 20 for 1 h. After 8-10 rinses wifla distilled water, the leaves were surface sterilized with 5% solution of liquid bleach v/v, (= 0.26 % w/v sodium hypochlorite) for 15 ~ followed by a thorough wash (x 5) with sterile distilled water. Leaf discs from sterilized leaves were then punched with the help of a sterile cork borer (5 ram) and inoculated on to solidified MS /Murashige and Skoog 1962) medium containing 3% sucrose (w/v), 0.8% agar (w/v) (Qualigens, bacteriological grade) ~md supplemented with various combinations of 2, 4-D and KN for callus induction. Leaf discs were cultured in 25 x 150 mm culture tubes containing 15 ml medium each. and aluminittm foil wraps were used to close the culture vessels. After 15 d of culttu-e, the proliferating calli were transferred to 150 ml Erlenmeyer flasks (Borosil) with 50 ml agar - solidified medium and maintained therein for 30 d. Callus initiation was visually assessed on a scale of 1-4, 1 being smallest mad 4 the largest. Scale 0 was used ffthere was no callus formalaon. Callus index was calculated as : ,,rn X G ~

N

x 100

172 ~uere

:

n = nttmber of callused leaf discs G = average callus rating as per visual scale (1-4) N = total number of cultured explants Subsequently,about 250 mg of the friable callus was transferredto 100 ml Erlenmeyer flasks with 30 tel MS liquid medium containing 1 mg/L 2, 4-D + 0.l mg/L KN. The cultures were agitated on a gyratory shaker at 100 rpm. After two weeks, larger cell clumps in the suspensionwere allowed to settle down and 100 ml aliquots of the superaantant,containing small cell clusters, were transferredto 50 ml of fi'esh medium The process was repeated every week for maintenmaceof embryogeniccell suspensions.

production of somatic embryos, which is in agreement with the report of Meijer and Brown (1987) in alfalfa. Of all the auxins used (NAA, IAA, 2, 4-D), 2, 4-D was the only auxin effective ha initiating embryogenic callus and development of embryoids. Table 1: Callus formation from young leaf tissue of ThevetiaperuviaT#a in response to various concentratians of 2, 4-D and kinetin MS

For routine observations,samplesof suspensionswere transferredto small dishes madobservedusing an invertedmicroscope (LeitzFLUOVERTFU). The embryogenic nature of cells was determined by staining with 2% (w/v) acetocarmine (Gupta and Durzxm 1987). Embryogenic cell clusters washed with MS basal medium were plated on semi - solid MS medium containing0.1 mg/L 2, 4-D ~mdvarious concentrationsofKN ~ad 2-iP. Calli with clustersof embryoidswere transferredto mediumwithoutgrowth regulators for expression of embryogenesis. Well-developed, isolated embryos were devellopedinto plantlets on modified MS media [ 1/2 MS with 2% sucrose, without NI:[~NOj and doubleKNOj (3800rag/L) ]. "Ihe pH of the cultttremediumwas adjustedto 5.8 prior to autoclavingat 121"C, 98 kPa for 20 miu. The cultures were incubated at 24 + 2 ~ C trader coolwhite lluorescentlight (40 u reel ma s-')with 16:g h light and dark photoperiod. Each treatment,except embryo development,con~sted of 20 replicates and the experimentwas repeated twice. Planters with 3-4 leaves and strong, viableroot systemswere trmasferredto earthen pots with autoclavedsoil : sand (1 : 1) mixture for acclimatization as described earher (Kumar t992b). RESULTS AND DISCUSSION

Callus proliferation was observed from the cut ends and epidermal surfaces of leaf discs, cultured on MS medium with 2, 4-D alone or m combination with K N Initially, maximum callus induction was observed on MS medium with 2 mg/L 2, 4-D + 0.1 mg/L K N with the whole disc covered with callus growth after 3 weeks (Table 1), However, callus proliferation decreased later and necrosis was observed. These calli, if broken into 2-3 m m pieces and cultured on MS medium with lower growth regadator levels of 1 mg/L 2, 4-D + 0.1 mg/L KN, revived their proliferating potential and growth rate. Calli varied in texture and organisation. Smooth, round structures resembling globular embryos were observed present in the calli, but these did not develop any further. Proliferation of caUi and presence of similar structures upon lowering growth regulator levels were also observed by Guimaraes et al (1988) in Cyphomandra. Though callus induction on the medium with 2 mg/L 2, 4-D + 0. It mg/L K N was faster than that on 1 mg/L 2, 4-D + 0.1 rag/ L KN, calli induced on the former stopped proliferation after 30 d and also lacked embryogenic competence when tested subsequently. Optimum proliferation of calli with embryogenie competence was observed on MS medium with 1 mg/L 2, 4-D + 0.1 mg/L K N (Table 1). Higher concentrations of 2, 4-D suppressed callus growth as well as embryogenic induction. Cultures subjected to 2, 4-D levels above 5 mg/L did not survive. Our observations suggest that higher 2, 4-D levels in the callus induction medium is necessary to enhance embryo induction, However, longer exposure to 2, 4t-D induction medium decreases the

2, 4-D + Kmetin (rag/L) 0+0 0§ 0.5 + 0 1.0 + 0 2.0 + 0 5.0+0 0,5 + 0.05 1.0 + 0,1 2.0 + 0.1 2,0 + 0.5 5,0 + 0.l

Callus index'

Embryogenic callus in&rationb

0 0 150 170 210 0 160 290 310 220 0

a calculatedas describedin the Materials and Methods b - negative, + Dositive One - month - old friable, greenish-white calli transferred to liquid MS medium with the same growth regulator complement, i.e. 1 mg/L 2, 4-1) + 0.1 mg/L KN and agitation on a gyratory shaker at 100 rpm resulted in development of cell suspensions, The proportion of small embryogenic cell clusters was gradually increased by subculmring the supematant after allowing the larger cell clumps to settle. After 4-6 transfers, each at 1 week intervals, these suspensions were freely dispersed and uniform, displaying groups of small, round, densely cytoplasmic, starch-containingceils with strong akNfity for acetocarmine. After 5-8 passages of selective subculturing, the suspension resulted m a more homogeneous culture manifesting only the early developmental stages of somatic embryogenesis. Further development and maturation of somatic embryos occurred when the suspension cultures were plated on MS medium containing quite a tow level (i.e. 0.1 rag/L) of 2, 4-D, and higher concentrations (1-2 rag/L) of cytokinins, KN and 2-iP, either individually or ha combinations (Table 2). The cell cultures treated with K N or 2-iP grew rapidly and produced embryoids of various stages (Fig. 1a). Percentage of calli showing somatic embryogenesis with 0.1 rag/ L 2, 4-D plus 2 mg/L K N or 2-iP was h~gh, with 2-iP manifesting highest frequency of embryogenic callus and number of embryoids/callus (Table 2). The use of cytokinins in the culture medium was important for embryo development. A smlilar effect of cytokinin supplementation on somatic embryogenesis was observed by Raghav Ram aud Nabors (1984) in rice cultures. It seems probable that Thevetia leaves have a high level of endogenous auxin and need exogenous cytokinins to promote embryo development.

173

Figures 1 (a-d) Somatic

embryogenesis

in

Thevetia

Fig.a.

Suspension derived callus showing somatic embryos on MS medium with 0.1 mg/L 2, 4-]3 and 2.0 mg/L 2 - iP

Fig.b.

Development of T. pemviana planflets from plated embryogenic callus on basal MS medium wiflaout growth regulators

Fig.c.

Complete planrlet recovered from development of somatic embryo on half-slxengthMS medium without NHNO~ and with double KNOx (3800 mg/L)

Fig.d.

Potted regenerant growing lmder the glass house

Alfllough most plants require an exogenous auxin for induction of somatic embryos (Carman 1990), the need for cytokinin during embryo development has been reported in only a few cases. In Coffee canephora, embryo development was inhibited by auxin, while the cytokmin had a beneficial effect on the process (Hatanaka et al 1991). Upon transfer to semi-solid MS medium lacking growth regulators, Thevetia cultures readily produced mature somatic embryos and complete plantlets (Fig. lb). The regenerauts were easily isolated and planted in pots for acclimatization. Isolated somatic embryos were capable of complete development on proper medium and gave rise to plantlets (Fig. lc),

unlike cases where embryo germination is difficult to achieve and growth is arrested at the eotyledonary stage (Tremouilloux- Guiller and Chenieux 1991) or due to secondary callusing (Mariotti and Arcioni 1983). Various modifications of MS medium were tried for embryo development into plants. Nearly 80% of isolated mature embryos plated on semi-solid half-strength MS salts medium + 2% sucrose with no N-H4 NO, and with double KNO x (3800 mg/L), formed complete, apparently normal plants. This medium or slightly varied ones (with B~ vitamins and 1% sucrose) have been used previously for embryo development (Finer 1988; Trolinder and Godwin 1987~ Umbeck et al 1987). Double nitrate medium has also been used for development of

174 Table 2 : Effect of kinetin and 2-iP on somatic embryogenesis (S.E.) from T. pemvimm callus cultures on MS medium with 0.1 mg/L

ACKNOWLEDGEMENTS

2, 4-D ~

Authors thank Head, Botany Department, NEHU, Shillong for laboratory facilities for carrying out part of the work and Director, FFPL for various courtesies extended.

Cytokinin Concenlration Number (rag/L) of calli tested Kinetin 2-iP 0 0.5 1.0 2.0 3.0 5.0 0 0 0 0 0 0.5 0.1 0.5 1.0

0 0 0 0 0 0 0.5 1.0 2.0 3.0 5.0 0.1 0.5 0.5 1.0

20 28 46 58 52 26 44 46 48 36 22 18 26 32 32

t~opol~ion of calli showing S.E. (%)

Embryoids/ callus clump *

0.0 53.5 60.8 84.4 48.0 15,3 54,5 73,9 87,5 61,1 31,8 44,4 38.4 37.5 65.6

0 42 + 4 42 + 8 55 + 4 54 + 6 35 +3 70 + 4 89 + 5 92 +_6 87 + 5 79 + 4 47 + 4 51 + 8 54 + 3 69 + 6

a data recorded after 45 d * 20 replicates/treatment; repeated twice; mean + standard ellor vigorous roots, better growth and high frequency regeneration in Leucaena leucocephala (Datta and Datta 1985). Plantlets recovered f r o m isolated embryoids had poorly developed roots and required a 2 w e e k dark incubation on half-strength M S m e d i u m with 2 % sucrose and no growth regulators for the d e v e l o p m e n t o f a proper root system. F r o m each 100 m g o f plated e m b r y o g e n i c callus, an average o f 80 plants were obtained within 3 months. O f the 60 plants subjected to acclimatization, 38 survived under glass house conditions (Fig. ld). All regenerated plants were green and normal, and appeared similar to plants in their natural environment. S o m e e m b r y o g e n i c tissue was maintained in a m e d i u m containing 2, 4-D for almost 2 years, and deleterious effect o f the long-term exposure have not been encountered a m o n g the primary regenerants. In general, e m b r y o g e n i c suspension cultures lose their regenerative capability with time. This report clearly demonstrates the initiation o f highly regenerable callus cultures from y o u n g leaf tissue o f mature Thevetia peruviana tree, the establishment o f long - term regenerable suspension culture and recovery o f normal plants from such suspensions. Besides producing large n u m b e r s o f somatic embryos capable o f f o r m i n g complete plants, the suspension culture study reported here can provide a useful system for protoplast to w h o l e plant regeneration because e m b r y o g e n i c cell suspensions have been s h o w n to be a superior source o f regenerable protoplasts in several species (Rhodes et al 1988).

REFERENCES

Baker CM, Wetzstein HY (1992) Plant Cell Reports 1l : 71-75 Bhakuni DS (1990).Sci Rep 8:12-17 Carman JO (1990) In Vilro Cell Dev Biol 26:746-753 Datta K, Datta SK (1985) Cttrr Sci 54:248-250 Eapen S, George L (1989) Plant Sci 61:127-130 Finer 33 (1988) Plant Cell Reports 7:399-402 Gupta PK, Durjan DJ (1987) Bio/Technology 5:147-151 Guimaraes MLS, Cruz GS, Montezuma-de-Carvatho JM (1988) Plant Cell Tiss Org Cult 15:161 - 167 Haccius B (1978) Phytomorphology 28:74-81 Hatanaka T, Arakawa O, Yasuda T, Uchida N, Yamaguchi T (1991) Plant Cell Reports 10:179-182 Kumar A (1992a) Ind J Exp Biol 30:749-750 Kumar A (1992b) Plant Cell Tiss Org Cult 3l: 47-50 Mariotti D, Arcieni S (1983) Plant Cell Tiss Org Cult 2:103-110 Meijer EG, Brown DC (1987) Physiol Plant 69:591-596 Murashige T, Skoog F (1962) Physiol Plant 15:473-497 Nagmani R, Beewar MR, Wenn SR (1987) Plant Cell Reports 6:157-159 Raghav Ram NV, Nabors MW (1984) Z Pflanzenphysiol 113: 315-323 Rhodes CA, Lowe KS, Ruby KL (1988) Bio/Technology 6:56-60 Trolinder NL, Godwin JR (1987) Plant Cell Reports 6:178-181 Tremonilloux-Gttiller J, Chenieux JC (1991) Plant Cell Reports I0:102-105 Umbeck P, Johnson G, Barton K, Swain W (1987) Bio/Technology 5: 263266 yon Amold S (1988) News Letter IAPTC 56:2-13